Agents for magnetic imaging method

ABSTRACT

The invention provides MRI detectable species of formula (I)
 
D p -S n -N m   (I)
 
wherein
         D is a MRI detectable moiety   S is a spacer   N is a molecule of a nutrient or pseudo-nutrient   n is 0 or an integer   m is an integer and   p is an integer.       

     These compounds are useful for internalizing into tumor cells an amount of the MRI detectable moiety that is distinguishably higher than the amount internalized in normal healthy cells thus allowing the diagnosis of tumors. 
     The internalization of the MRI detectable moiety involves the nutrients or pseudo-nutrients transporting system. Preferred compounds of formula (I) are those wherein D is the chelated complex of a paramagnetic metal ion.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.10/516,781, filed Dec. 12, 2004, which is the national stage filing ofcorresponding international application number PCT/EP03/05761, filedJun. 2, 2003, which claims priority of the European application no. EP02012531.6, filed Jun. 5, 2002, all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to new MRI detectable species suitable foruse as contrast agents in a magnetic imaging method, and to theinjectable compositions containing them for the magnetic resonanceimaging (MRI) of living subjects and for the diagnosing of tumors.

The invention also refers to the injectable compositions of some of saidnew MRI detectable species for the therapy of tumors.

Finally the present invention also relates to the new intermediates inthe preparation of the new MRI detectable species of the invention.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI) is a nuclear magnetic resonancetechnique that is used clinically to distinguish between differenttissues or organs in the human or animal body through the spatiallocalization of water protons in the tissues or organs. The signal thatis obtained by this technique and is then converted into an imagingdepends in fact on the water proton concentrations and on the relaxationrates within the different types of tissues.

MRI often requires the use of contrast agents, i.e. agents thatinfluence the local relaxation behaviour of the observed nuclei incertain tissues or organs, because if MRI is performed without employinga contrast agent, differentiation of the tissue of interest from thesurrounding tissues in the resulting image may be difficult.

The in vivo utilization of paramagnetic complexes as non-specific agentsfor contrast enhanced MRI has been the subject of a number of differentstudies. Paramagnetic contrast agents involve materials which containunpaired electrons. The unpaired electrons act as small magnets withinthe main magnetic field to increase the rate of longitudinal (T₁) andtransverse (T₂) relaxation. Generally, paramagnetic contrast agents areused for their ability to decrease T₁ (positive contrast agents) and inuse they enhance image intensity from the regions to which theydistribute.

Paramagnetic contrast agents typically comprise metal ions, such as forinstance transition metal ions, which provide a source of unpairedelectrons. Particularly preferred and therefore studied in more depth,resulted to be Gd³⁺ (with 7 unpaired electrons) and Mn²⁺ (with 5unpaired electrons). Particular attention has been paid to the Gd³⁺ ionas this ion shows a very high magnetic moment coupled to a relaxationrate relatively long at the magnetic fields of interest in the MR area(in the range of nanoseconds). However these metal ions are alsogenerally highly toxic or at least poorly tolerated and need to bestrongly coordinated with a ligand that occupies the major number ofcoordination sites. Generally speaking, to control toxicity and at thesame time get a sufficient contrast in the imaging, it is necessary tohave paramagnetic complexes endowed with high thermodynamic and kineticstability, containing at least one molecule of water directlycoordinated to the metal ion in rapid exchange with the “bulk” water.

The first contrast agent for MRI approved by the Regulatory Authoritieswas GdDTPA (Magnevist®, by Schering AG), followed by GdDOTA (Dotarem®,by Guerbet SA), GdDTPA-BMA (Omniscan®, by Nycomed Imaging AS), andGdHPDO3A (ProHance®, by Bracco Imaging S.p.A.). The chemical formula ofthese contrast agents is reported hereinbelow

These four contrast agents share some similar pharmacological featuresas they all diffuse from plasma into the extracellular fluids and areexcreted through the kidney via glomerular filtration. They areparticularly useful for the diagnosis of hematoencephalic barrierlesions.

Linked thereto are other two Gd(III) complexes that are used in theimaging of the liver: Gd EOB-DTPA (Eovist®, by Shering AG) and Gd BOPTA(MultiHance®, by Bracco Imaging S.p.A.) (the chemical formula of the twoligands, EOB-DTPA and BOPTA, is reported below)

These two compounds are characterised by an increased lipophilicbehaviour due to the introduction of an aromatic substituent in theligand structure and for this reason are preferably uptaken by the livercells.

Another class of compounds useful as contrast agents for MRI areferromagnetic materials which are employed for their ability to decreaseT₂. Ferromagnetic materials have high, positive magneticsusceptibilities and maintain their magnetism in the absence of anapplied field. Ferromagnetic materials for use as MRI contrast agentsare for instance described in WO 86/01112 and WO 85/043301.

A third class of magnetic materials that can be used in MRI are thosegenerally indicated as superparamagnetic materials. Analogously toparamagnetic materials, the superparamagnetic ones do not maintain theirmagnetism in the absence of an externally applied magnetic field.Superparamagnetic materials can have magnetic susceptibilities nearly ashigh as ferromagnetic materials and higher than the paramagnetic ones.As generally used, also superparamagnetic materials alter the MR imageby decreasing T₂ and therefore result in a darkening of the tissues orfluids where they are present or accumulate versus the lighterbackground where they are not present.

Iron oxides such as magnetite and gamma ferric oxide exhibitferrromagnetism or superparamagnetism depending on the size of thecrystals comprising the material, with larger crystals (typically withan average size larger than 0.3 μm) being ferromagnetic.

The general idea of an MRI enhancing contrast agent comprising a moietythat is detectable in a magnetic resonance imaging procedure linked to amolecule capable of specific binding to a cellular receptor is alreadyknown.

See for instance U.S. Pat. No. 4,827,945 that discloses i.e. coatedmagnetite particles for use as MRI contrast agents, said particles beingsurrounded by a polymer to which biologically active molecules, chosento target specific organs or tissues, may be attached.

See also WO 01/30398 and the literature cited therein where the use ofreceptor-binding ligands bound to a paramagnetic chelate is described.

The idea is that since there are specific receptors which are known tobe overexpressed in the cells of certain tumors, being able toselectively distinguish a tumor cell from a normal cell will allow tovisualize and identify precise locations of the tumor masses and bettermanage the disease.

The paramagnetic, ferromagnetic or superparamagnetic compoundscontaining the suitably selected targeting moiety (e.g. antibody,antibody fragment, peptide, protein, and the like) bind to the relevantreceptors on the surface of the tumor cells to be targeted and ispossibly internalised. The number of receptors per cell however isgenerally lower than the number of MRI detectable metal ions required tohave a MR signal visible with the actual MRI technologies. To increasethe contrast and make the signal more visible it is therefore necessaryto increase the concentration of the contrast agent in the cell or onthe surface of the cell. This can be obtained either giving to the cellsufficient time to internalize the label/targeting compound (so that thesignal can be given by the contrast agent inside the cell as well asaround or on the surface of the cell) and/or administeringsimultaneously a compound capable to increase internalization of thereceptor-binding compound, or increasing the number of MRI detectablemetal units linked to the targeting moiety (via dendrimers ormultimers). In the latter case, particularly useful is theinternalization route based on receptor mediated (fluid) endocytosisthat allows the entrapment of a huge number of paramagnetic units.

While theoretically excellent, in practice neither of these approacheshas led to a commercial product yet. On the one hand, in fact, once thetargeting MRI detectable compound is bound to the cell surface, it isnot always possible to achieve or induce the desired internalization andon the other hand the use of large multimers is generally connected withan unacceptable increase in toxicity of the product because of the largemolecular weight thus obtained.

For utility in diagnostic imaging the optimum contrast agent shouldprovide a contrast sufficient to clearly distinguish between normal,healthy cells and tumor cells using the available equipment and withoutcreating any toxicity problem.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides new compounds suitable foruse in the manufacture of contrast agents, said compounds containing anMRI detectable moiety and being functionalised in such a way to beeasily internalised into the cells.

More particularly the present invention provides for a MRI detectablespecies that is useful for internalising into tumor cells an amount of aMRI detectable metal that is distinguishably higher than the amountinternalised in normal healthy cells.

It has now been found in fact that it is possible to distinguish betweentumor cells and normal cells using contrast agents based on a MRIdetectable species that contain at least one MRI detectable moiety thatis bound, either directly or through a spacer, to at least one moleculeof a nutrient or pseudo-nutrient. The nutrient or pseudo-nutrienttransporters or transporting systems that are present into the humanbody, will in fact recognize the nutrient or pseudo-nutrient moleculeand will carry it, together with the at least one MRI detectable moietythat is linked thereto, into the cells where said MRI detectable moietyor moieties will thus concentrate to provide a more evidentvisualization of the cell. The altered metabolism of the tumor cellsthat require a much higher amount of nutrients or pseudo-nutrients orthat selectively employ certain specific pseudo-nutrients, will allow aclear distinction between tumor and normal cells as the former ones willinternalise a much higher amount of the compounds containing a MRIdetectable moiety.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising an effective amount of a MRI detectable speciesof the invention together with at least one pharmaceutically acceptablecarrier. In a preferred embodiment said pharmaceutical composition willbe in the form of an injectable composition.

Said injectable composition can be employed for diagnostic purpose usingthe MRI technology to visualize tumor cells and, when in the newcompounds the MRI detectable moiety is a chelated complex of aparamagnetic metal.

In still a further aspect the present invention also relates to aprocess for the preparation of the new compounds by conjugating the MRIdetectable moiety D or a precursor thereof with the nutrient orpseudo-nutrient molecule, either directly or through a spacer, and tothe new intermediates obtained therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 reports the differential uptake of the compound of Example 1 by ahuman hepatoma cell line with respect to hepatocytes

FIG. 2 reports the differential uptake of the compound of Example 3 by ahuman hepatoma cell line with respect to hepatocytes

FIG. 3 summarises the differential uptake of the compound of Example 3by various cancer cell lines.

FIG. 4 reports the differential uptake of the compound of Example 8 byrat hepatocytes with respect to human hepatoma cells HTC.

FIG. 5 reports the differential uptake of the compound of Example 8 in apanel of cancer cell lines with respect to healthy hepatocytes and 3T3cells.

FIG. 6 reports the differential uptake of the compound of Example 8 inHTC cells as function of the concentration of glutamine.

DETAILED DESCRIPTION OF THE INVENTION

More particularly a first object of the present invention is a MRIdetectable species of following formula (I)D_(p)-S_(n)N_(m)  (I)

wherein

-   -   D is a MRI detectable moiety    -   S is a spacer    -   N is a molecule of a nutrient or pseudo-nutrient    -   n is 0 or an integer    -   m is an integer and    -   p is an integer.

The MRI detectable species of formula (I) must contain at least one MRIdetectable moiety D and at least one nutrient or pseudo-nutrientmolecule N bound together.

If in the above formula (I) m is 1 and p is 1, then D and N can be boundtogether either directly (n=0) or through a spacer S (n=1).

It is also possible to have, in the above formula (I), one MRIdetectable moiety D bound to more than one nutrient or pseudo-nutrientmolecule N, either directly or through a number of spacers S which maybe up to the number of nutrient or pseudo-nutrient molecules N (i.e.,p=1, m>1, n=0 or an integer≦m). In such a case typically the number ofnutrient or pseudo-nutrient molecules will be up to 5, preferably up to4, more preferably up to 3 and even more preferably up to 2.

The MRI detectable species (I) may also contain more than one, andtypically up to 10, preferably up to 8, more preferably up to 6, andeven more preferably up to 4, MRI detectable moieties D, that can belinked to one molecule of nutrient or pseudo-nutrient N, either directlyor through one or up to an even number of spacers S (i.e., p>1, n≦p,m=1). In case the more than one MRI detectable moieties D are linked tothe N molecule through one spacer S, said spacer will preferably containa multiplicity of binding sites equal to the number of MRI detectablemoieties D, i.e., the spacer S will provide the backbone on whichseveral D moieties or a cluster of D moieties are bound.

When the MRI detectable species of formula (I) contains more than oneMRI detectable moiety D, said moieties can also be bound to more thanone nutrient or pseudo-nutrient molecule N through a spacer S containinga multiplicity of binding sites (i.e., p>1, n=1, m>1.

In the formula (I) above therefore p typically will be an integer offrom 1 to 10, preferably of from 1 to 8, more preferably of from 1 to 6,and even more preferably of from 1 to 4, i.e., 1, 2, 3 or 4; n istypically 0 or an integer of from 1 to 5, preferably 0, or an integer offrom 1 to 3, more preferably 0 or an integer of from 1 to 2, and evenmore preferably 0 or 1; and m is typically an integer of from 1 to 5,preferably of from 1 to 4, more preferably of from 1 to 3, and even morepreferably 1 or 2.

While generally the MRI detectable species of the present invention canbe represented as a characterizable compound of formula (I) as indicatedabove, there may be instances where the MRI detectable species accordingto the present invention can only be represented as a combination ofmoieties (D and N and optionally S) bonded or otherwise associated, e.g.conjugated, with each other. As used herein however the term “MRIdetectable species of formula (I)” will include also these compositionsof matter.

In formula (I) above D is any MRI detectable moiety, i.e. any moietywhich affects local electromagnetic fields (i.e. any paramagnetic,superparamagnetic or ferromagnetic species), that contains at least oneportion capable of being linked to at least one molecule of nutrient orpseudo-nutrient N, either directly or through a spacer S.

The MRI detectable moiety D therefore can be a coated ferromagneticparticle, a coated superparamagnetic particle or a chelated complex of aparamagnetic metal ion wherein the coating of the ferromagnetic orsuperparamagnetic particles or the chelator of the paramagnetic metalcontain at least one site for a possible link to a spacer S or to anutrient/pseudo-nutrient molecule N.

Examples of suitably coated ferromagnetic or superparamagnetic particlesare for instance those described in U.S. Pat. Nos. 4,770,183, 4,827,945,5,707,877, 6,123,920, and 6,207,134 where the coating materials, i.e.,polymers such as polysaccharides, carbohydrates, polypeptides,oreanosilanes, proteins, and the like, gelatin-aminodextran, or starchand polyalkylene oxides, can be functionalised to allow binding of theparticle to the spacer or to the nutrient/pseudo-nutrient molecule.

In a preferred embodiment of the present invention however the MRIdetectable moiety D is a paramagnetic metal ion complexed with achelating ligand L.

Preferred paramagnetic metal ions include ions of transition andlanthanide metals (i.e. metals having atomic number of 21 to 29, 42, 43,44, or 57 to 71). In particular ions of Mn, Fe, Co, Ni, Eu, Gd, Dy, Tm,and Yb are preferred, with those of Mn, Fe, Eu, Gd, and Dy being morepreferred and Gd being the most preferred.

As known in the art and used herein, the term “chelator” or “chelatingligand” is intended to refer to a compound containing donor atoms thatcan combine by coordinative bonding with a metal atom to form a cyclicstructure (coordination cage) called “chelation complex” or “chelate”.

Suitable chelating ligands L that are or can be functionalised in such away to allow binding of the paramagnetic chelation complex D to thespacer or nutrient/pseudo-nutrient molecule as in formula (I) above,include the residue of a polyaminopolycarboxylic acid, either linear orcyclic, in racemic or optically active form, such asethylenediaminotetracetic acid (EDTA), diethylenetriaminopentaaceticacid (DTPA),N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethyl]-L-glycine(EOB-DTPA), N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-L-glutamic acid(DTPA-GLU),N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutamine,N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-L-lysine (DTPA-LYS), the DTPAmono- or bis-amide derivatives, such asN,N-bis[2-[carboxymethyl[(methylcarbamoyl)methyl]amino]ethyl]glycine(DTPA-BMA),4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,1,1-triazamidecan-13-oicacid (BOPTA), 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid(DOTA), 1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid (DO3A),10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid(HPDO3A), 2-methyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraaceticacid (MCTA);(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraaceticacid (DOTMA),3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triaceticacid (PCTA),[(4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid or of a derivative thereof wherein one or more of the carboxylicgroups are in the form of the corresponding salts, esters, or amides; orof a corresponding compound wherein one or more of the carboxylic groupsis replaced by a phosphonic and/or phosphinic group, such as forinstance4-carboxy-5,11-bis(carboxymethyl)-1-phenyl-12-[(phenylmethoxy)methyl]-8-(phosphonomethyl)-2-oxa-5,8,11-triazamidecan-13-oicacid,N,N′-[(phosphonomethylimino)di-2,1-ethanediyl]bis[N-(carboxymethyl)glycine],N,N′-[(phosphonomethylimino)di-2,1-ethanediyl]bis[N-(phosphonomethyl)glycine],N,N′-[(phosphinomethylimino)di-2,1-ethanediyl]bis[N-(carboxymethyl)glycine],1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylen(methylphosphonic)]acid,or1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylen(methylphosphinic)]acid.

Suitable chelating ligands L as well as the processes for theirpreparation are described for instance in the following patents:GB-A-2,137,612, EP-A-230,893, EP-A-255,471, EP-A-299,795, EP-A-325,762,EP-A-565,930, EP-A-594,734, U.S. Pat. No. 4,885,363, EP-A-773,936,WO-A-9426313, WO-A-9426754, WO-A-9426755, WO-A-9519347, WO-A-9731005,WO-A-9805625. WO-A-9805626, WO-A-9935133, WO-A-9935134, andWO-A-0146207, which are incorporated herein by reference.

Preferred chelating ligands L are linear and macrocyclicpolyaminopolycarboxylic acids, in racemic or optically active form.

More preferred are ethylenediaminotetracetic acid (EDTA),diethylenetriaminopentaacetic acid (DTPA),N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethyl]-L-glycine(EOB-DTPA), N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-L-glutamic acid(DTPA-GLU), N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-L-lysine(DTPA-LYS),N,N-bis[2-[carboxymethyl[(methylcarbamoyl)methyl]amino]ethyl]glycine(DTPA-BMA),4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazamidecan-13-oicacid (BOPTA), 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid(DOTA), 1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid (DO3A),10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid(HPDO3A), 2-methyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraaceticacid (MCTA),(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraaceticacid (DOTMA),3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triaceticacid (PCTA),[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid and the derivatives thereof wherein one or more of the carboxylicgroups are in the form of the corresponding alkali metal, alkaline earthmetal, or quaternary ammonium salts, (C₁-C₄)alkyl esters orunsubstituted, mono- or di-substituted amides.

Most preferred are ethylenediaminotetracetic acid (EDTA),diethylenetriaminopentaacetic acid (DTPA),4-carboxy-5,8,1-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazamidecan-13-oicacid (BOPTA), 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid(DOTA), 1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid (DO3A), and10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecan-1,4,7-triacetic acid(HPDO3A),(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraaceticacid (DOTMA), as well as the analogs thereof where the methyl groups arereplaced by higher alkyl homologs,3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triaceticacid (PCTA) and[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid.

As used herein the term “nutrient” refers to any substance or compoundthat is essential for sustaining the cell life in the organismconcerned, while the term “pseudo-nutrient” refers to any substance orcompound that is nonessential during health but is required in the dietin certain pathophysiologic states because cell utilisation exceeds thecapacity for endogenous biosynthesis or to any substance or compoundthat is capable of being utilised by the cells in any of their vitalfunctions or as an assimilable source of nutrients.

Examples of “nutrients” are typically monosaccharides and the essentialamino acids. The term “monosaccharides” includes glucose, which is themost preferred one, fructose and other hexoses. The term “essentialamino-acid” generally refers to the L-isomers of the α-amino acids whichare found in nature.

Typical examples of “pseudo-nutrients” are the polyamines, thederivatives of the essential amino acids and the non-essentialamino-acids.

Examples of polyamines are e.g. putrescine, spermidine, and spermine,that are present in all the cells and play an important role in severalessential cell functions through their interaction with DNA, RNA,proteins, and lipids. The uptake of these compounds by the rapidlygrowing cells as well as by the tumor cells is known to be very high.

A suitable example of essential amino-acid derivative suitable aspseudo-nutrients is e.g. agmatine, the compound obtainable bydecarboxylation of arginine, that can be used by the cell as a nitrogensource and is known to be internalised by the cells through the sametransport system used for polyamines.

Non essential amino-acids are e.g. the D-isomers of the naturalessential amino-acids, any mixture of the L- and D-isomers, as well assynthetic α-amino acids which are not found in nature. An example ofmolecule suitable as pseudo-nutrient is glutamine that is not anessential amino-acid but becomes a conditionally essential amino acid inthe host with cancer. Glutamine in fact is known to be the main sourceof nitrogen for tumor cells that compete with the host for circulatingglutamine.

Other substances or compounds not specifically listed above may howeverbe employed as the nutrient or pseudo-nutrient molecule. Moreparticularly any substance or compound that is recognized by at leastone transporting system of the living organism that receives the MRIdetectable species (I) and that is internalised by the tumor cells in ahigher amount with respect to the normal healthy cells, can suitably beemployed.

Since there are several different transporting systems that are known topreferentially transport one or the other nutrient or pseudo-nutrientmolecule into the cell of a given tissue or organ, it would thus bepossible also to specifically design the type of nutrient orpseudo-nutrient to be coupled with the MRI detectable moiety D in such away to preferentially direct the MRI detectable species (I) to thetarget cells.

Examples of known transporting systems are for instance the ASC systemthat is expressed by different types of cells, that typically transportsneutral apolar or neutral polar amino-acids, such as alanine, leucine,valine, methionine, serine, cysteine, glutamine, threonine, asparagine;the N system that specifically transports amino-acids that containsnitrogen in the side chain, such as glutamine, asparagine and histidine;the B^(0.+) system that transports also cationic amino-acids and isexpressed only in certain organs, i.e. lung and trachea; the Y⁺ systemthat is ubiquitous with the only exception of epatocytes and mediatesthe uptake of cationic amino-acids as well as that of glutamine; the Lsystem for the transport of neutral apolar amino-acids, such as leucine,isoleucine, valine, phenylalanine, tyrosine, tryptophane, methionine,and of some neutral polar amino-acids, such as glutamine, serine andthreonine; the T system that preferentially transports neutral aromaticamino-acids; and the various families of glucose transporters, GLUT1,GLUT2, GLUT3, GLUT4, and GLUT5, which are expressed in the varioustissues and are responsible for the cellular uptake of glucose and theother hexoses.

When more than one molecule of nutrient or pseudo-nutrient N is present,each N can be independently selected among the nutrient andpseudo-nutrient molecules as indicated above. In a preferred embodimenthowever when more than one molecule N is linked to the MRI detectablemoiety D, all the N molecules are of the same compound or substance.

As indicated above, in the case of nutrients, saccharides and essentialamino acids are the preferred molecules, wherein the most preferred onesare glucose, neutral amino acids such as alanine and phenylalanine, andcationic amino acids such as lysine and arginine.

In case of pseudo-nutrients, polyamines such as putrescine andspermidine, non essential amino-acids, such as glutamine and essentialamino acid derivatives such as agmatine are the most preferredcompounds.

Each of the molecules of nutrient or pseudo-nutrient N can be linked tothe MRI detectable moiety D either directly or through a spacer S.

When the MRI detectable moiety D is linked to a nutrient orpseudo-nutrient N through a direct bond, said bond usually involvesinteraction between functional groups located on the nutrient molecule Nand on the MRI detectable moiety, i.e. on the coating of theferromagnetic or superparamagnetic particle or—in the preferredembodiment of the present invention according to which D is aparamagnetic chelated complex—on the chelating ligand L. In general, nonlimitative examples of chemically reactive functional groups which maybe employed to this purpose include amino, hydroxy, thiol, carboxy,carbonyl and the like groups.

In the preferred embodiment where the MRI detectable moiety D is aparamagnetic metal ion complexed with a chelating ligand L, thechelating ligand L may be attached directly to the nutrient orpseudo-nutrient molecule N via one of the metal coordinating groups ofthe chelant which may form an ester, amide, thioester, or thioamide bondwith an amine, thiol, or hydroxy group present on the N molecule. Insuch a case preferably the ligand L will contain free carboxyl groups,and a direct covalent bond with the nutrient or pseudo-nutrient moleculeN can be obtained through formation of an ester or, preferably, an amidebond with respectively a hydroxy or amino group possibly present in theN molecule. Alternatively, or additionally, the N molecule and thechelating ligand L may be directly linked via a functionality attachedto the chelant L backbone, and in such a case it is also possible todevise a ligand L bearing as a substituent in its backbone e.g. theresidue of an amino-acid or amino-acid derivative, a polyamine, or aglucose molecule N.

Alternatively to the direct joining of the MRI detectable moiety D tothe nutrient or pseudo-nutrient molecule N, these two elements can alsobe connected through a homo- or hetero-bifunctional linker, i.e. aspacer S. In this case said spacer S will contain at least two specificreactive moieties separated by a spacing arm, wherein one of thereactive moieties will provide for a covalent bonding with the MRIdetectable moiety D and the other with the nutrient or pseudo-nutrientmolecule N. The spacing arm may typically consist of an alkylidene,alkenylidene, alkynylidene, cycloalkylidene, arylidene, or aralkylideneradical that can be substituted and be interrupted by heteroatoms suchas oxygen, nitrogen, and sulphur. In a preferred embodiment said spacerarm consists of an aliphatic, straight or branched chain, thateffectively separates the reactive moieties of the spacer so thatideally the spatial configuration of the molecule of nutrient orpseudo-nutrient N is not influenced by the presence of the MRIdetectable moiety D and the molecule of nutrient or pseudo-nutrient N isthus more easily recognized by the transporter. Said chain may beinterrupted by groups such as, —O—, —S—, —CO—, —NR—, —CS— and the likegroups or by aromatic rings such as phenylene radicals, and may bearsubstituents such as —OR—, —SR, —NRR₁, —COOR, —CONRR₁, and the likesubstituents, wherein R and R₁, each independently, may be a hydrogenatom or an organic group.

The reactive moieties in said bifunctional spacer, that may be the sameor, preferably, different, need to be capable of reaction with thefunctional groups present in the MRI detectable moiety D and in thenutrient or pseudo-nutrient molecule N, i.e. need to be able to reactwith carboxyl, amino, hydroxyl, sulphydryl, carbonyl, and the likegroups.

Examples of functional groups capable of reaction with carboxylic groupsinclude diazo compounds such as diazoacetate esters and diazoacetamides,which react with high specificity to generate ester groups. Carboxylicacid modifying reagents such as carbodiimides, e.g.1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (CMC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), may also beusefully employed. Other useful carboxylic acid modifying reagentsinclude isoxazolium derivatives, chloroformates, andN-carbalkoxydihydroquinolines. Examples of reactive moieties capable ofreaction with sulphydryl groups include α-haloacetyl compounds andmaleimide derivatives. Examples of reactive moieties capable of reactionwith amino groups include alkylating and acylating agents.Representative of the alkylating agents are α-haloacetyl compounds,maleimide derivatives, reactive aryl halides and alkyl halides,α-haloalkyl ethers, aldehydes and ketones capable of Schiff's baseformation with amino groups (the adducts formed usually being stabilisedthrough reduction to give a stable amine), epoxide derivatives, such asepichlorohydrin and bisoxiranes. Examples of acylating agents includeisocyanates and isothiocyanates, acid anhydrides, acid halides, activeesters, and those useful reagents for amide bond formation widely knownand conventionally used for peptide syntheses.

Said spacer may also contain more than two functional groups. Inparticular when a single spacer molecule S is used to bind more than oneMRI detectable moiety D to one or more than one nutrient orpseudo-nutrient molecule N or vice-versa, said spacer may contain amultiplicity of possible binding sites or be a molecular aggregate witha multiplicity of built-in or pendant groups which bind covalently ornon-covalently (e.g. coordinatively) with the MRI detectable moieties Dand the nutrient or pseudo-nutrient molecules N in such a way to anchorsaid moieties thereto with a strength and for a time sufficient to bringthe MRI detectable species (I) into the cells.

The nature of the spacer S may have a critical bearing on the stabilityof the end MRI detectable species and on the capability for thetransporting systems to recognize the nutrient or pseudo-nutrientmolecule. As indicated in fact it should bind the MRI detectable moietyD to the recognized nutrient or pseudo-nutrient molecule N for anadequate period of time that would allow the MRI detectable moiety D tobe internalised by the tumor cells in an amount sufficient to give aclearly distinguishable contrast imaging. It should also bind the MRIdetectable moiety D to the nutrient or pseudo-nutrient molecule in a waythat would ensure an appropriate spatial conformation that allows thenutrient or pseudo-nutrient molecule to be easily recognized by thetransport system. In a preferred embodiment it should also bebiodegradable, i.e. contain a bond that may be susceptible to anenzymatic or a chemical cleavage, particularly once it has beentransported into the cell. Furthermore, when according to the preferredembodiment of the invention the MRI detectable moiety D is aparamagnetic metal chelated complex, said optional spacer S should alsoensure that water molecules may have access to the chelated paramagneticion (as the entity of the signal will depend on the rate of exchange ofthe water molecule of the coordination cage with the water bulk).

When more than one molecule of spacer S is present, each of them can beindependently selected as indicated above. According to a preferredembodiment, however, in such a case all the n molecules of spacer S areidentical.

The reaction conditions used for obtaining the MRI detectable species(I) will vary depending on the particular type of reactive moietiesemployed but are analogous to those known in the literature for similargeneral reactions and can be easily devised by any skilled technician.

In general, for the preparation of the MRI detectable species (I), it ispossible to conjugate first the spacer, if any, with the nutrient orpseudo-nutrient molecules N and then conjugate the obtained intermediateproduct with the MRI detectable moiety or moieties D; or alternatively,it is also possible to conjugate first the MRI detectable moiety ormoieties D with the spacer or spacers, if any, and then conjugate theobtained intermediate with the nutrient or pseudo-nutrient molecule(s)N. However, when in the MRI detectable species of formula (I), D is aparamagnetic metal chelated complex, preferably the process for thepreparation of these compounds will comprise conjugating one or morechelating ligands L to one or more nutrient or pseudo-nutrient moleculesN either directly or through one or more spacers S, by any suitablesequence of steps, to get a suitable precursor of the desired endcompound (I), said precursor having following general formula (II)L_(p)-S_(n)-N_(m)  (II)

wherein

-   -   L is a chelating ligand    -   S is a spacer    -   N is a molecule of a nutrient or pseudo-nutrient    -   n is 0 or an integer    -   m is an integer and    -   p is an integer.

and then metallating the chelant groups L in said intermediate compound(II) with the suitably selected paramagnetic metal. The precursors,intermediate compounds, of formula (II), that will also be indicatedherein as “functionalised chelators”, represent a further specificobject of the present invention. In the above formula (II) the meaningsof L, S, N, n, m, and p are as defined above in relation with the endcompounds (I). Further objects of the present invention are thefollowing preferred functionalized chelators, according to generalformula (II), as well as the processes for their preparation thereof.6,16-dicarbonyl-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandiguadinine,6,16-dicarbonyl-5,19-dicarboxy-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandioicacid diamide, 3,6,9-triaza-3,6,9-tricarboxymethylundecanoic acidbis-glucopyranosylamide,2,24-diamino-8,18-dicarbonyl-7,10,13,16,19-pentaaza-10,13,16-tricarboxymethyl-pentaheicosandioicacid,2,16-dibenzyl-4,13-dicarbonyl-3,6,9,12,15-pentaaza-6,9,12-tricarboxymethyl-heptadecandioicacid,10,20-dicarbonyl-4,9,12,15,18,21,26-heptaaza-12,15,18-tricarboxymethyl-nonaheicosan-1,29-diamine,4,26-diamino-5,10,20,25-tetracarbonyl-12,15,18-tricarboxymethyl-6,9,12,15,18,21,24-heptaaza-nonaheicosan-1,29-diguanidina;N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutamine,N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-agmatine,N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-arginine and[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmetl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid.

Most preferred areN,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutamine,N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-agmatine,N,N-Bis[2-[bis(carboxymethyl)amino)ethyl]-L-γ-glutamyl-arginine and14-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid.

However, a further preferred embodiment of the present invention are thecorresponding complexes compounds, according to general formula (I), ofthe above indicated list of compounds. In particular with ions of Mn,Fe, Co, Ni, Eu, Gd, Dy, Tm, and Yb are preferred, with those of Mn, Fe,Eu, Gd, and Dy and Gd being more preferred and Gd being the mostpreferred.

In particular the persons skilled in the art are well aware of the factthat is highly preferred to work with chelates of high thermodynamicstability in order to limit potential toxic effects associated to therelease of free metal ions.

Among the methods generally known for incorporating a metal ion into achelator, i.e. direct incorporation, template synthesis andtransmetallation, the direct incorporation is the preferred one toobtain the compounds of formula (I) wherein D is a paramagnetic metalchelated complex starting from the intermediates of formula (II). Thusthe MRI detectable species (I) wherein D is a paramagnetic chelatedcomplex can be easily obtained by merely exposing or mixing an aqueoussolution of the functionalized chelators (II) with a paramagnetic metalsalt in an aqueous solution having a pH comprised between about 4 andabout 9, and preferably comprised between about 5 and about 8. The saltcan be any salt but preferably a water soluble salt of the paramagneticmetal, such as a halide. Said salts are preferably selected so as not tointerfere with the binding of the metal ion with the functionalisedchelator (II). The functionalised chelator (II) is preferably in aqueoussolution at a pH of between about 5 and about 8. It can be mixed withbuffer salts such as citrate, acetate, phosphate and borate, to producethe optimum pH. Preferred paramagnetic metal ions include ions oftransition and lanthanide metals (i.e. metals having atomic number of 21to 29, 42, 43, 44, or 57 to 71). In particular ions of Mn, Fe, Co, Ni,Eu, Gd, Dy, Tm, and Yb are preferred, with those of Mn, Fe, Eu, Gd, andDy being more preferred and Gd being the most preferred.

Preferably the MRI detectable species (I) wherein D is a paramagneticchelated complex should have a zero residual charge on the coordinationcage in order not to alter the residual charges possibly present on thenutrient or pseudo-nutrient moiety of the end species (I).

Preferred cations of inorganic bases suitable for salifying theparamagnetic chelated complexes, if necessary, comprise the ions ofalkali metals or alkaline-earth metals such as potassium, sodium,magnesium, calcium, and the like metals, including any mixed salt.Preferred cations of organic bases suitable for this purpose comprisethose obtained by protonation of primary, secondary and tertiary amines,such as ethanolamine, diethanolamine, morpholine, glucamine,N-methylglucamine, N,N-dimethylglucamine, basic amino-acids such aslysine, arginine, and ornithine, and the like organic bases.

The MRI detectable species (I) wherein D is a paramagnetic chelationcomplex, should contain at least one molecule of water in thecoordination cage of the chelation complex so as to guarantee a rlphigher than 1 s⁻¹mM⁻¹. Higher relaxivities (such as higher than 4 s⁻¹mM⁻¹, or higher than 8 or higher than 10 s⁻¹mM⁻¹ or higher than 15 s⁻¹mM⁻¹) can be advantageous but not strictly necessary because of the highamount of internalized MRI detectable moiety D.

The MRI detectable species (I) according to the present invention may beadministered to patients for imaging in an amount sufficient to give thedesired contrast with the particular technique used in the MRI.Generally, dosages of from about 0.001 to about 5.0 mmoles of MRIdetectable species (I) per kg of body weight are sufficient to obtainthe desired contrast. For most MRI applications preferred dosages ofimaging metal compound will be in the range of from 0.01 to 2.5 mmolesper kg of body weight.

The MM detectable species (I) of the present invention can be employedfor the manufacture of a contrast medium for use in a method ofdiagnosis by MRI involving administering said contrast medium to a humanor animal being and generating an image of at least part of said humanor animal being.

More particularly the MRI detectable species (I) according to thepresent invention can be employed for the manufacture of a contrastmedium for use in a method of diagnosing tumors, said method involvingadministering said contrast medium to a human or animal being anddetecting the major uptake of said detectable species by tumor cellswith respect to normal cells.

For said use the MRI detectable species (I) of the present invention maybe formulated with conventional pharmaceutical aids, such asemulsifiers, stabilisers, anti-oxidant agents, osmolality adjustingagents, buffers, pH adjusting agents, and the like agents, and may be ina form suitable for parenteral administration, e.g. for infusion orinjection.

Thus the MRI detectable species (I) according to the present inventionmay be in conventional administration forms such as solutions,suspensions, or dispersions in physiologically acceptable carriersmedia, such as water for injection.

Parenterally administrable forms, e.g. i.v. solutions, should be sterileand free from physiologically unacceptable agents, and have lowosmolality to minimize irritation and other adverse effects uponadministration. These parenterally administrable solutions can beprepared as customarily done with injectable solutions. They may containadditives, such as anti-oxidants, stabilizers, buffers, etc., which arecompatible with the chemical structure of the MRI detectable species (I)and which will not interfere with the manufacture, storage and usethereof.

Said pharmaceutical compositions may also contain suitable agents thatmay increase the amount of MRI detectable moiety internalised. As anexample it would be possible to administer the MRI detectable species(I) in combination with a compound that specifically inhibits theenzymes responsible for the biosynthesis of the nutrient orpseudo-nutrient N within the cell. This would lead to an increaseduptake of the exogenous nutrient N in the form of MRI detectable species(I). Said “internalization adjuvant” may be administered simultaneouslywith the MRI detectable species (I) and in such a case the formulationmay contain both ingredients. Alternatively it can be administered inadvance with respect to the MRI detectable species (I) and in such acase two separate formulations should be provided that may be presentedin the form of a kit.

The following examples further illustrate the present invention in somerepresentative embodiments thereof and in particular describe thepreparation of some representative compounds of formula (I) andintermediates of formula (II) according to the present invention. Aswill be known to those of skill in the art, however, these samecompounds may also be prepared following different synthetic routes.

Example 1 Preparation of the Gadolinium Complex of6,16-dicarbonyl-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandiguadinineGadolinium Complex ofN,N′-(4-guanidinobutyl)diethylenetriaminopentaacetic acid bis-amide

a) Preparation of diethylenetriaminopentaacetic acid bis-anhydride

Diethylenetriaminopentaacetic acid (10 g; 0.0255 mol) and pyridine(14.54 ml; 0.18 mol) are charged into a 100-ml reaction flask equippedwith magnetic stirrer, heating oil bath, and dripping funnel. While thetemperature is kept at the room value and the solution is stirred,acetic anhydride (10.56 ml; 0.11 mol) is added dropwise. The reactionmixture is heated to 65° C. for 3 hours, and then cooled to roomtemperature. The solid obtained is recovered by filtration on büchner,washed on filter with acetic anhydride (2×10 ml), methylene chloride(2×10 ml) and ethyl ether (2×10 ml). The white powder is then driedunder vacuum yielding 8.82 g of the compound of title a) (97%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of6,16-dicarbonyl-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandiguadinine

A mixture of agmatine free base (3.27 g; 0.02531 mol) and DMSO (70 ml)is stirred at 50° C. for two hours. A solution of the compound obtainedin step a) above (4.20 g; 0.01175 mol) in dimethylsulfoxide (DMSO) (15ml) is added portionwise over a period of two hours and the mixture isallowed to react at 50° C. for 4 hours and then at room temperature foradditional 20 hours. At the end of the reaction time the solution isclear but a lower yellow phase of a jelly consistency is present. TheDMSO phase is recovered by decantation and acetone is added theretountil precipitation of a solid product is complete. The lower yellowphase is dissolved in a small amount of water and acetone is addedthereto to get an additional crop of solid precipitate. The two cropsare combined and washed several times with fresh acetone, left understirring overnight. The surnatant is then removed and the solid is takenup with water, washed again with acetone, and dried yielding 8.03 g of araw product that is dissolved in acid water (pH=2) and purified bycolumn chromatography on Amberlite™ XAD 1180.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c) Preparation of the Gadolinium Complex of6,16-dicarbonyl-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandiguadinine

An aqueous solution of the chelating ligand obtained in step b) above(7.0 g; 0.0122 mol) is brought to pH 6.5 by the addition of NaOH and anequimolar amount of GdCl₃ (3.22 g; 0.0122 mol) is then slowly stirred inat room temperature. During the addition the pH of the reaction mixtureis monitored and adjusted to 6.5 with NaOH. Once the addition of GdCl₃is over, the solution is brought to pH 8.5 and filtered on a 22 μmsyringe filter. A small amount of the chelating ligand obtained in stepb) above is then added to the filtrate and the pH is brought to 7.Removal of the solvent under vacuum yields the compound of the title asa white solid.

Example 2 Preparation of the Gadolinium Complex of6,16-dicarbonyl-5,19-dicarboxy-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandioicacid diamide Gadolinium Complex ofN,N′-glutamin-diethylenetriaminopentaacetic acid bis-amide

a) Preparation of6,16-dicarbonyl-5,19-dicarboxy-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandioicacid diamide

A solution of L-glutamine (5.00 g; 0.0342 mol) in water (80 ml) isloaded into a 100-ml reaction flask and sodium hydroxide (1.37 g; 0.0342mol) is added thereto. While keeping the reaction temperature at 15-20°C., diethylenetriaminopentaacetic acid bis-anhydride obtained asdescribed in step a) of Example 1 (6.11 g; 0.0171 mol) is addedportionwise and the reaction mixture is stirred under nitrogenatmosphere for 4 hours. The pH of the reaction mixture is thenneutralised by the addition of HCl and the solvent is evaporated offunder reduced pressure yielding a white product that is purified bycolumn chromatography on Amberlite™ XAD 1180 at pH 2 eluting with water.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of the Gadolinium Complex of6,16-dicarbonyl-5,19-dicarboxy-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandioicacid diamide

The gadolinium complex of the chelating ligand of step b) above has beenprepared by following the same general procedure described in step c) ofExample 1.

Example 3 Preparation of the Gadolinium Complex of3,6,9-triaza-3,6,9-tricarboxymethylundecanoic acidbis-glucopyranosylamide

Gadolinium complex of N,N′-glucosamin-diethylenetriaminopentaacetic acidbis-amide a) Preparation of3,6,9-triaza-3,6,9-tricarboxymethylundecanoic acidbis-glucopyranosylamide

A solution of glucosamine (1.00 g; 0.00463 mol) in DMSO (10 ml) ischarged into a 100-ml reaction flask and kept under stirring at 40° C. Asolution of diethylenetriaminopentaacetic acid bis-anhydride obtained asdescribed in step a) of Example 1 (0.746 g; 0.00209 mol) in DMSO (5 ml)is slowly added thereto and the reaction mixture is stirred undernitrogen atmosphere for 4 hours. Methanolic KOH is added to bring the pHof the reaction mixture to 8 and the reaction product is thenprecipitated by addition of methanol followed by the addition ofacetone. The precipitate is washed several times with fresh acetone toremove DMSO still present and the product is then dried under vacuum,Yield 64%.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of the gadolinium complex of3,6,9-triaza-3,6,9-tricarboxymethylundecanoic acidbis-glucopyranosylamide

The gadolinium complex of the chelating ligand of step b) above has beenprepared by following the same general procedure described in step c) ofExample 1.

Example 4 Preparation of the Gadolinium complex of2,24-diamino-8,18-dicarbonyl-7,10,13,16,19-pentaaza-10,13,16-tricarboxymethyl-pentaheicosandioicacid Gadolinium Complex ofN,N′-(5-amino-5-carboxy-pentyl)diethylenetriaminopentaacetic acidbis-amide

a)2,24-diamino-8,18-dicarbonyl-7,10,13,16,19-pentaaza-10,13,16-tricarboxymethyl-pentaheicosandioicacid

N-tertbutoxycarbonyl-L-lysine (0.950 g; 0.00386 mol) and DMSO (10 ml)are charged into a 100-ml reaction flask and stirred at 50° C. untilcomplete solution. A solution of diethylenetriaminopentaacetic acidbis-anhydride obtained as described in step a) of Example 1 (0.690 g;0.00193 mol) in DMSO (5 ml) is slowly added thereto over a period ofabout one hour. After about 24 hours methanolic KOH is added to bringthe pH of the reaction mixture to 8 and then acetone is added toprecipitate the product which is then washed several times with freshacetone. Upon removal of the solvent the obtained solid is dried at 40°C. in an oven yielding 1.05 g (64%) of the N,N′-tertbutoxycarbonylderivative of the compound of the title. Deprotection to give thecompound of the title is then achieved by stirring for 96 hours thesolid in diethyl ether containing 1M HCl (6 ml). A pale yellow solid isthus obtained.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of the gadolinium complex of2,24-diamino-8,18-dicarbonyl-7,10,13,16,19-pentaaza-10,13,16-tricarboxymethyl-pentaheicosandioicacid

The gadolinium complex of the chelating ligand of step b) above has beenprepared by following the same general procedure described in step c) ofExample 1.

Example 5 Preparation of the Gadolinium Complex of2,16-dibenzyl-4,13-dicarbonyl-3,6,9,12,15-pentaaza-6,9,12-tricarboxymethyl-heptadecandioicacid Gadolinium Complex ofN,N′-phenylalanyl-diethylenetriaminopentaacetic acid bis-amide

a) Preparation of2,16-dibenzyl-4,13-dicarbonyl-3,6,9,12,15-pentaaza-6,9,12-tricarboxymethyl-heptadecandioic acid

A solution of phenyalanine (0.66 g; 0.002 mole) in DMSO (10 ml) isloaded into a 100-ml reaction flask and the mixture is heated to 50° C.under stirring. A solution of diethylenetriaminopentaacetic acidbis-anhydride obtained as described in step a) of Example 1 (0.715 g;0.002 mol) in DMSO (6 ml) is slowly added thereto over a period of abouttwo hour. The reaction mixture is allowed to react at 50° C. for 4 hoursand at room temperature for additional 20 hours. At the end of this timethe solution is clear but a lower yellow phase of a jelly consistency ispresent. The DMSO phase is recovered by decantation and acetone is addedthereto until precipitation of a solid product is complete. The loweryellow phase is dissolved in a small amount of water and acetone isadded thereto to get an additional crop of solid precipitate. The twocrops are combined and washed several times under stirring with freshacetone. The surnatant is then removed and the solid is taken up withwater and washed again with fresh acetone. The raw material thusobtained is dissolved in acidic water (pH=2) and purified by columnchromatography on Amberlite™ XAD 1180.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of the Gadolinium Complex of2,16-dibenzyl-4,13-dicarbonyl-3,6,9,12,15-pentaaza-6,9,12-tricarboxymethyl-heptadecandioicacid

The gadolinium complex of the chelating ligand of step b) above has beenprepared by following the same general procedure described in step c) ofExample 1.

Example 6 Preparation of the Gadolinium Complex of10,20-dicarbonyl-4,9,12,15,18,21,26-heptaaza-12,15,18-tricarboxymethyl-nonaheicosan-1,29-diamineGadolinium Complex of N,N′-spermidin-diethylentriaminopentaneacetic acidbis-amide

a) Preparation of10,20-dicarbonyl-4,9,12,15,18,21,26-heptaaza-12,15,18-tricarboxy-methyl-nonaheicosan-1,29-diamine

A solution of diethylenetriaminopentaacetic acid bis-anhydride obtainedas described in step a) of Example 1 (0.357 g; 0.001 mol) in DMSO (5 ml)is added over a period of two hours to a solution ofN-(3-aminopropyl)-1,4-butanediamine (spermidine, 1.45 g; 0.01 mol) inDMSO (20 ml) stirred at 50° C. in a 100-ml reaction flask. The reactionmixture is allowed to react at 50° C. for 4 hours and then at roomtemperature for additional 20 hours. At the end of this time thesolution is clear but a lower yellow phase of a jelly consistency ispresent. The DMSO phase is recovered by decantation and acetone is addedthereto until precipitation of a solid product is complete. The loweryellow phase is dissolved in a small amount of water and acetone isadded thereto to get an additional crop of solid precipitate. The twocrops are combined and washed several times under stirring with freshacetone. The surnatant is then removed and the solid is taken up withwater and washed again with fresh acetone. The raw material thusobtained is dissolved in acidic water (pH=2) and purified by columnchromatography on Amberlite™ XAD 1180.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of the gadolinium complex of10,20-dicarbonyl-4,9,12,15,18,21,26-heptaaza-12,15,18-tricarboxymethyl-nonaheicosan-1,29-diamine

The gadolinium complex of the chelating ligand of step b) above has beenprepared by following the same general procedure described in step c) ofExample 1.

Example 7 Preparation of the Gadolinium Complex of4,26-diamino-5,10,20,25-tetracarbonyl-12,15,18-tricarboxymethyl-6,9,12,15,18,21,24-heptaaza-nonaheicosan-1,29-diguanidinaGadolinium Complex ofN,N′-arginylethylendiammino-diethylentriaminopentaneacetic acidbis-amide

a) Preparation of4,26-diamino-5,10,20,25-tetracarbonyl-12,15,18-tricarboxymethyl-6,9,12,15,18,21,24-heptaaza-nonaheicosan-1,29-diguanidina

2-amino-5-guanidino-valeric acid (arginine, 0.871 g; 0.005 mol) isdissolved in an aqueous solution at pH 9 and a strong excess of benzylchloroformate is then slowly added thereto. The thus obtainedcorresponding tri-carbobenzyloxy derivative is isolated, dissolved inmethylene chloride and added with di-cyclohexylcarbodiimide. Theobtained solution is then slowly added to a solution of ethylenediamine(1.5 g; 0.025 mol) in methylene chloride. The 2-aminoethylamide of thetri-carbobenzyloxy protected arginine is recovered, dissolved in DMSOand a solution of diethylentriaminopentaneacetic acid bis-anhydride(1.78 g; 0.005 mol) in DMSO is then added to the obtained solutionheated to 50° C. The reaction mixture is stirred at this temperature for4 hours and then cooled to room temperature. The pH is adjusted to 8 bythe addition of methanolic KOH and the raw product that precipitates iswashed with acetone under stirring overnight. The solid is thenrecovered, dissolved in methanol and the protecting carbobenzyloxygroups are removed by catalytic hydrogenation over Pd/C 5%. Any residualcarbamic acid that may possibly be present is then removed byacidification with HCl yielding the desired compound.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of the Gadolinium Complex of4,26-diamino-5,10,20,25-tetracarbonyl-12,15,18-tricarboxymethyl-6,9,12,15,18,21,24-heptaaza-nonaheicosan-1,29-diguanidina

The gadolinium complex of the chelating ligand of step b) above has beenprepared by following the same general procedure described in step c) ofExample 1.

Example 8 Preparation of[[N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutaminato(6-)gadolinate(3-)]trisodiumsalt Gadolinium Complex ofN,N-Bis[2-ibis(carboxymethyl)amino]ethyl]-L-γ-glutaminyl-L-glutamine

a) Preparation ofN,N-Bis[2-[bis(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-γ-glutamyl-L-glutaminebis(1,1-dimethylethyl)ester

A solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDCI) (1.93 g; 10.00 mmol) in CH₂Cl₂ was added over 20min to a solution ofN,N-bis[2-[bis(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid 1-(1,1-dimethylethyl)ester (6.25 g; 8.38 mmol) (i) andN-hydroxysuccinimide (NHS) (1.16 g; 10.05 mmol) in CH₂Cl₂ stirred at0-5° C. After 24 h at room temperature the reaction solution was washedwith H₂O, dried (Na₂SO₄) and evaporated to give the activated esterwhich was dissolved in CH₂Cl₂. This solution was added dropwise over 30min to a solution of L-glutamine (1,1-dimethylethyl) ester hydrochloride(2 g; 8.38 mmol) (ii) and triethylamine (1.27 g; 12.55 mmol) in CH₂Cl₂at room temperature. After 3 days at room temperature the solution waswashed with H₂O and dried (Na₂SO₄). The solution was evaporated to givea crude (7.77 g) that was purified by flash-chromatography (iii) to give3.94 g (4.24 mmol) as a pale yellow oil. Yield 51%.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation ofN,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutamine

Trifluoroacetic acid (12.4 mL; 162.3 mmol) was added to a solution ofthe compound obtained in the step a) (5.03 g; 5.4 mmol) in CH₂Cl₂ (100mL) and stirred at room temperature for 48 h. The solvent was evaporatedand the residue was dissolved in trifluoroacetic acid (10 mL). After 48h at room temperature the acid was evaporated and the crude was treatedwith Et₂O, then filtered. The solid was suspended twice in Et₂O andfiltered to give a white solid (3.76 g, 4.1 mmol). Yield 75%.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c) Preparation of[[N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutaminato(6-)]gadolinate(3-)]trisodiumsalt

A solution of the compound obtained in the step b) (2.77 g; 3.00 mmol)in H₂O was adjusted to pH 7.7 by addition of 2 N NaOH (5 mL); Gd₂O₃(1.03 g; 2.80 mmol) was added and the suspension was stirred at 50° C.for 24 h. As the pH was 12, 1 N HCl (2 mL) was added to reach the valueof 7. The excess Gd₂O₃ was filtered off (0.43 g; 1.20 mmol) and thesolvent was evaporated to give the product as a pale yellow solid (3.10g; 2.95 mmol). Yield 98%.

Example 9 Preparation of[[N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl(5-)-agmatine]gadolinate(3-)]disodiumsalt Gadolinium Complex ofN,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-agmatine)

a) Preparation ofN,N-Bis[2[bis(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-γ-glutamyl-agmatine

N-hydroxysuccinimide (1.41 g; 12.29 mmol) was added to a solution ofN,N-bis[2-[bis(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid 1-(1,1-dimethylethyl) ester (7.64 g; 10.24 mmol) in CH₂Cl₂ (100mL). The mixture was cooled at 0° C. and a solution of EDCI (2.36 g;12.29 mmol) in CH₂Cl₂ (45 mL) was added dropwise. The mixture wasstirred at room temperature, monitoring the reaction by TLC. When thestarting material was completely consumed, the solution was washed withwater (3×100 mL), dried (Na₂SO₄) and evaporated. The residue (9.28 g)was dissolved in DMF (120 mL) and agmatine (1.19 g; 10.24 mmol) wassuspended in the solution. The mixture was stirred at room temperaturefor 2 d. The solution was filtered and water (150 mL) was added. Thesolution was washed with Et₂O and the evaporated organic phase wastreated with brine (100 mL). The aqueous phase was extracted with Et₂Oand organic phase was dried and evaporated. The residue was purified byflash chromatography to give the compound (1.71 g; 1.99 mmol) as yellownon-crystalline solid. Yield 19%

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation ofN,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-agmatine

The compound obtained ad indicated in step a) (1.48 g; 1.72 mmol) wasdissolved in CH₂Cl₂ (15 mL) and TFA (3.3 mL; 43 mmol) was added to thesolution. The mixture was stirred at room temperature. After 4 days thesolvent was removed under reduce pressure to give 1.56 g of chideproduct. The raw materials was dissolved in pure TFA (5 mL) and themixture was stirred at room temperature overnight. TFA was removed underreduced pressure and the residue was crystallized with Et₂O and filteredto give a white crystalline solid. Because of the presence of Et₂O andTFA in the final product, the solid was dissolved in water again andevaporated under reduce pressure for three times. The final product wasobtained as white crystalline solid (0.77 g; 1.33 mmol). Yield: 97%.

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c) Preparation of[[N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl(5-)-agmatine]gadolinate(3-)]disodium salt

The compound as obtained in the previous step b) was dissolved in 10 mLof water and NaOH 1M was added (2.3 mL). Then NaOH 0.1M was slowly addeduntil the pH of final solution was 7. Gd₂O₃ (210 mg; 0.58 mmol) wassuspended and the mixture was heated at 50° C. overnight. Because of pHof the solution raised to 11.8, HCl 1M (0.6 mL) was added to adjust thepH to 5. The mixture was maintained at room temperature for further 48 hthen filtered (Millipore HA 0.45) to eliminate the unreacted Gd₂O₃. ThepH of the solution was adjusted to 6.4 and the solvent was evaporatedunder reduced pressure to give 0.95 g (1.22 mmol) of white crystallinesolid.

Example 10 Preparation of[[N,N-Bis[2[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl(5-)-arginine]gadolinate(3-)]disodium salt Gadolinium Complex ofN,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-arginine

a) Preparation ofN,N-bis[2-[bis(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-γ-glutamyl[2-amino-5-[N′[(2,3,6-trimethyl-4-methoxy)benzensulfonyl]]guanidine-1-(1,1-dimethylethyl)ester]-L-glutamicacid 1-(1,1-dimethylethyl)ester

N-hydroxysuccinimide (773 mg; 6.72 mmol) was added to a solution ofN,N-bis[2-[bis(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid 1-(1,1-dimethylethyl)ester (4.18 g; 5.6 mmol) in CH₂Cl₂ (55 mL).The mixture was cooled at 0° C. and a solution of EDCI (1.29 g; 6.72mmol) in CH₂Cl₂ (25 mL) was added dropwise. The mixture was stirred atroom temperature, monitoring the reaction by TLC. When the startingmaterial was completely consumed, the solution was washed with water(3×60 mL), dried (Na₂SO₄) and evaporated. The residue (4.81 g) wasdissolved in CH₂Cl₂ (55 mL) and2-amino-5-[N′[(2,3,6-trimethyl-4-methoxy)benzensulfonyl]]guanidine-1-(1,1-dimethylethyl)ester(2.5 g; 5.6 mmol) was added to the solution. The mixture was stirred atroom temperature for 25 h. The solution was washed with water (3×50 mL)and the organic phase was dried and evaporated. The residue was purifiedby flash chromatography to give the product (5.45 g; 4.66 mmol) as whitecrystalline solid. Yield 83%

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation ofN,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-arginine

The compound obtained in step a) (2.5 g; 2.14 mmol) was dissolved in amixture of TFA (63 mL) and thioanisole (7 mL). The solution was stirredat room temperature. After 2 days the solvent was removed under reducepressure and the residue was dissolved in HCl N (18 mL) and evaporated;this procedure was repeated three times. The residue was dissolved inwater and extracted with CHCl₃. The aqueous solution was evaporated toobtained a crude (1.79 g) that was dissolved in water (6 mL), adjustedto pH 1.7 by addition of NaOH 1M and purified by percolation throughAmberlite XAD 1600T resin column to give the product (1.03 g; 1.66 mmol)as white crystalline solid. Yield: 78%

c) Preparation of[[N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl(5-)-arginine]gadolinate(3-)]disodiumsalt

The compound as obtained in the step b) was dissolved in water (15 mL)and NaOH 1M was added (2.5 mL) until the pH of the solution was 3.9 (0.5mL). Gd₂O₃ (217 mg; 0.6 mmol) was added and the solution was heated to50° C. overnight. Because of pH of the solution raised to 12.5, HCl 1M(0.35 mL) was added to adjust the pH to 6.5. The mixture was maintainedat room temperature for further 3 days, then it was filtered (MilliporeHA 0.45 filter) to eliminate the unreacted Gd₂O₃. The pH was adjusted to6.90 and the solvent was evaporated under reduced pressure to give 1.08g (1.32 mmol) of white crystalline solid.

Example 11 Preparation of Gadolinium Complex of[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid

a) Preparation of 3-[(4-Aminobutyl)amino]propanenitrile

Acrylonitrile (5.08 g, 0.096 mol) was added to ice bath-cooled1,4-butanediamine (8.44 g, 0.096 mol) in 30 min. The resulting mixturewas stirred for 2 h at RT, heated to 50° C. for further 3 h and finallylet stirring at RT overnight. After Kugelrohr distillation colourlessoil was obtained (5.80 g, 0.041 mol, yield 42.8%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

b) Preparation of tert-ButylN-{4-{[(tert-Butyloxy)carbonyl]amino}butyl}-N-(2-cyanoetyl)carbamate

BOC—ON (9.41 g, 0.038 mol) and was added in small portions to a solutionof 3-[(4-Aminobutyl)amino]propanenitrile (2.70 g, 0.019 mol) andtrietylamine (6.00 g, 0.059 mot) in dioxane/H₂O (9:1 v/v, 50 cm³). Theresulting mixture was stirred at room temperature for 3 days; then thesolvent was removed under reduced pressure. The resulting pale yellowoil was dissolved in Et₂O (80 cm³) and washed with 1M NaOH (3×30 cm³)and brine (2×25 cm³). The organic fraction was dried over Na₂SO₄ andfiltered. After removal of the solvent, a pale yellow oil was obtainedwhich was dried under reduced pressure (6.041 g, 0.0177 mol, yield92.7%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

c) Preparation of tert-ButylN-(3-aminopropyl)-N-{4-{[(tert-Butyloxy)carbonyl]amino}butyl}carbamate

The product as obtained in step b) (2.07 g, 6.066 mmol) and NaOH (0.6 g,0.015 mol) were dissolved in 94% EtOH (30 ml). After addition ofNi-Raney as catalyst, the resulting mixture was hydrogenated underpressure (10 bar). After filtration through celite, the solvent volumewas reduced to 5 cm³ and the product was extracted with CHCl₃ (5×50cm³). The collected organic fractions were dried over Na₂SO₄ andfiltered. After removal of the solvent, a pale yellow oil was obtainedwhich was dried under reduced pressure (1.826 g, 5.29 mmol, yield87.2%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

d) Preparation of1,6-bis-(ten-Butyloxycarbonyl)-11-p-nitrophenyl-1,6,10-triazaundecane

To a solution of the compound as obtained in step c) (1.64 g, 4.30 mmol)in Et₂O (30 cm³) 4-nitrobenzaldehyde (0.716 g, 4.30 mmol) dissolved in amixture of Et₂O and THF (20 cm³, 1:1 v/v) was added in 1 h. Theresulting mixture was stirred at RT for 24 h and then the solvent wasremoved at the rotary evaporator. The reddish oil obtained was dissolvedin EtOH (30 cm³) and NaBH, (0.488 g, 12.9 mmol) was added in smallportions. The resulting solution was stirred at RT for 5 h. Afterremoval of the solvent the product was recovered with H₂O, basified with1M NaOH and extracted with CH₂Cl₂ (5×50 cm³). The collected organicfractions were dried over Na₂SO₄ and filtered to yield a yellow oilwhich was purified by column chromatography (from EtOAc 100% toEtOAc/MeOH 95:5) (0.550 g, 1.146 mmol, yield 26.6%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

e) Preparation of1,6-bis-(tert-Butyloxycarbonyl)-11-p-aminophenyl-1,6,10-triazaundecane

A mixture of the compound obtained in step d) (0.183 g, 0.382 mmol) inEtOH (20 cm³) and of FeO(OH) as catalyst (20 mg) was heated to 60° C.under N₂ atmosphere. Then hydrazine hydrate (19.5 mg, 0.60 mmol) wasadded and the resulting mixture was stirred 18 h at RT. After filtrationthrough celite the solvent was removed at the rotary evaporator to yielda yellow oil which was dried under reduced pressure. (0.112 g, 0.248mmol, yield 65.0%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

f) Preparation of1,6-bis-(tert-Butyloxycarbonyl)-11-(4-bromoacetamidophenyl)-1,6,10-triazaundecane

To a suspension of K₂CO₃ (304.0 mg, 2.2 mmol) and of the productobtained in the previous step e) (100.0 mg, 0.22 mmol) in dry CH₃CN (15cm³) cooled to 0° C. and kept under N₂ atmosphere, a solution ofBromoacetyl bromide (42.4 mg, 0.22 mmol) in dry CH₃CN (10 cm³) was addedin 1 h. After 3 h the mixture was filtered and the solvent was removedat the rotary evaporator to yield a yellow oil which was dried underreduced pressure (99.4 mg, 0.174 mmol, yield 79.0%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

g) Preparation of[4-(1,6-bis-(tert-Butyloxycarbonyl)-1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid tris-tert-butylester

To a suspension of K₂CO₃ (215.0 mg, 2.2 mmol) and of DO3AtBu (103.6 mg,0.174 mmol) in dry CH₃CN (15 cm³) heated to 80° C. and kept under N₂atmosphere, a solution of the compound obtained in step 1) (99.4 mg,0.174 mmol) in dry CH₃CN (10 cm³) was added in 1 h. After 18 h themixture was filtered and the solvent was removed at the rotaryevaporator to yield a brown oil which was dried under reduced pressure(100.4 mg, 0.10 mmol, yield 57.4%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

h) Preparation of[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid

The compound obtained in step g) (100.4 mg, 0.10 mmol) was dissolved ina mixture CH₂Cl₂/TFA (1:1, v/v, 20 cm³) and stirred at RT for 4 h afterwhich a reddish oil was formed. After decantation of the solution, MeOH(10 cm³) was added and a yellow solid was formed. The solid was thencollected and dried under reduced pressure (45.4 mg, 0.071 mmol, yield71.3%).

The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with the indicatedstructure.

i) Preparation of the gadolinium complex of[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid

The gadolinium complex of the chelating ligand of step h) above has beenprepared by following the same general procedure described in step c) ofExample 1.

The possibility of using the compounds of the present invention todistinguish tumor cells from normal healthy cells, thus allowing the useof the contrast agents based thereon in the diagnosis of tumors, hasbeen confirmed by evaluating the significantly increased rate ofinternalization of representative MRI detectable species (I) in variouscells lines including tumor cells and comparatively normal healthycells.

The experiments done and the results obtained are summarisedhereinbelow.

Example 12

Analysis of the differential uptake of the compound of Example 1 in rathepatocytes and human hepatoma cells HepG2, has been carried out asfollows:

Rat hepatocytes were cultured in M199 medium supplemented with 2 mg/mlbovine serum albumin (BSA), 3.6 mg/ml Hepes, 100 U/ml penicillin, 100U/μg, streptomycin, and 10 nmol/l insulin; HEPG2 cells were cultured inRPMI 1640 medium supplemented with 10% foetal bovine serum, 100 U/mlpenicillin and 100 U/μg streptomycin.

The uptake experiments were performed in 5 ml EBSS, Earl's Balanced SaltSolution, (CaCl₂ 0.266 g/l; KCl 0.4 g/l; NaCl 6.8 g/l; glucose 1 g/l;MgSO₄ 0.204 g/l; NaH₂PO₄ 0.144 g/l; NaHCO₃ 2.2 g/l).

Determination of Gd moles internalized was performed with the measure ofrelaxation rate at 20 MHz of cytosolic extracts after mineralizationovernight at 120° C. in 37% HCl (1:1). The value has been normalised on1 mg protein of the lisate of cells.

A time course analysis was done where cells were incubated at 37° C. inEBSS and then collected at different times (15′, 30′, 1 h, 2 h, 6 h).

The substrate concentration was for both cell lines 0.8 mM.

The results obtained show that after 1 hour the uptake of the compoundof Example 1 by the human hepatoma cells HepG2 is 3 times more that theone in hepatocytes, and at 2 hours is more than 4 times higher, reachinga plateau after about two hours and a half. These results aregraphically reported in FIG. 1.

Example 13 Differential Uptake of the Compound of Example 3 inHepatocytes and Hepatoma Cell Lines HepG2

The cells were cultured as in Example 12 above. The uptake experimentswere performed in 5 ml EBSS without glucose to avoid competition betweenthis compound and the test compound.

Determination of Gd moles internalized was performed with the measure ofrelaxation rate at 20 MHz of cytosolic extracts after mineralization at120° C. in 37% HCl (1:1). The value was normalised on 1 mg protein ofthe lisate of cells.

The substrate concentration was 5.1 mM and a time course analysis wasdone.

The results that show the higher uptake of the compound of Example 3 inhuman hepatoma cells HepG2 compared with hepatocyte are reported in FIG.2.

Example 14

The uptake of the compound of Example 3 in a panel of cancer cell linescompared with healthy hepatocytes was evaluated by culturing the variouscancer cell lines and performing the uptake experiments as describedabove.

Determination of the Gd moles internalized was performed with themeasure of relaxation rate at 20 MHz of cytosolic extracts aftermineralization at 120° C. in 37% HCl (1:1). The value was normalised on1 mg protein of the lisate of cells.

The substrate concentration was 5.1 mM and the uptake was measured after2 hours from the administration of the test compound.

The uptake of Gd was increased in all the cancer cell lines with a valuethat is cell line dependent. These results are summarised in thehistogram of FIG. 3.

Example 15 Analysis of the Differential Uptake of the Compound ofExample 8 in Rat Hepatocytes and Human Hepatoma Cells HTC

This experiment has been carried out as follows:

Rat hepatocytes were cultured in M199 medium supplemented with 2 mg/mlbovine serum albumin (BSA), 3.6 mg/ml Hepes, 100 U/ml penicillin, 100U/μg streptomycin, and 10 nmol/insulin; HTC cells were cultured in RPMI1640 medium supplemented with 10% foetal bovine serum, 100 U/mlpenicillin and 100 U/μg streptomycin.

The uptake experiments were performed in 5 ml EBSS, Earl's Balanced SaltSolution, (CaCl₂ 0.266 g/l; KCl 0.4 g/l; NaCl 6.8 g/l; glucose 1 g/l;MgSO₄ 0.204 g/l; NaH₂PO₄ 0.144 g/l; NaHCO₃ 2.2 g/l).

Determination of Gd moles internalized was performed with the measure ofrelaxation rate at 20 MHz of cytosolic extracts after mineralizationovernight at 120° C. in 37% HCl (1:1). The value has been normalised on1 mg protein of the lisate of cells.

A time course analysis was done where cells were incubated at 37° C. inEBSS and then collected at different times (1 h, 3 h, 6 h). Thesubstrate concentration was for both cell lines 1.3 mM. The resultsobtained show that after 0.5 hour the uptake of the compound of Example11 by the human hepatoma cells IITC is ⅓ times more that the one inhepatocytes, and at 2 hours is more than 3 times higher, reaching avalue of more than 4 times higher after six hours. These results aregraphically reported in FIG. 4.

Example 16

The uptake of the compound of Example 8 in a panel of cancer cell linescompared with healthy hepatocytes and 3T3 cell was evaluated byculturing the various cancer cell lines and performing the uptakeexperiments as described above.

Determination of the Gd moles internalized was performed with themeasure of relaxation rate at 20 MHz of cytosolic extracts aftermineralization at 120° C. in 37% HCl (1:1). The value was normalised on1 mg protein of the lisate of cells.

The substrate concentration was 1.6 mM and the uptake was measured after6 hours from the administration of the test compound.

The uptake of Gd was increased in all the cancer cell lines with a valuethat is cell line dependent. These results are summarised in thehistogram of FIG. 5.

Example 17

The uptake of the compound of example 8 in HTC cells as a function ofthe concentration of glutamine in the inoculation medium was evaluatedby culturing the cancer cell lines and performing the uptake experimentsas described above.

Determination of the Gd moles internalized was performed with themeasure of relaxation rate at 20 MHz of cytosolic extracts aftermineralization at 120° C. in 37% HCl (1:1). The value was normalised on1 mg protein of the lisate of cells.

The substrate concentration was 1.3 mM and the uptake was measured after1, 2 and 10 hours from the administration of the test compound. Cellswere incubated at 37° C. for 6 hours with a fixed amount of compound ofexample 8 and increasing concentration of the competitor glutamine (from0.5 to 10 mM). These results are summarised in the histogram of FIG. 6.

It is worth to recall that a general indication to access theinvolvement of a given transporter in the internalization of a given1-structure can be drawn by a competitive assay with the nutrient orpseudo-nutrient of choice.

While this invention has been described in reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompassany such modifications or embodiments.

The invention claimed is:
 1. An intermediate compound selected from thegroup consisting of:6,16-dicarbonyl-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandiguadinine;6,16-dicarbonyl-5,19-dicarboxy-5,8,11,14,17-pentaaza-8,11,14-tricarboxymethyl-heneicosandoicacid diamide;10,20-dicarbonyl-4,9,12,15,18,21,26-heptaaza-12,15,18-tricarboxymethyl-nonaheicosan-1,29-diamine;and4,26-diamino-5,10,20,25-tetracarbonyl-12,15,18-tricarboxymethyl-6,9,12,15,18,21,24-heptaaza-nonaheicosan-1,29-diguanidina.2. An intermediate compound selected from the group consisting of:N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-agmatine;N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-arginine;N,N-Bis[2-[bis(carboxymethyl)amino]ethyl]-L-γ-glutamyl-L-glutamine; and[4-(1,6,10-triazaundecan)-phenyl-aminocarbonylmethyl]-1,4,7,10-tetraazacyclododecan-4,7,10-triaceticacid.